Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard

Definitions

• the invention relates to standard control reference materials for use in cytokine assays.
• the invention also relates to methods for producing such materials.
• cytokine release by ELISPOT assay and the quantification and identification of intracellular cytokines by flow cytometry are both widely used as indices of cell mediated immune responses.
• cytokines released from immobilised cells typically interact with an immunoassay, such as an ELISA assay, to produce a coloured "spot" on the assay plate providing both qualitative (e.g. type of immune protein) and quantitative (number or proportion of cells responding) information.
• an immunoassay such as an ELISA assay
• Stimulated (cytokine positive), fixed, cryopreserved cells are available commercially for intracellular flow cytometry (Becton Dickinson, Oxford, UK), and freeze dried unstimulated cells are available either as controls for surface staining or for purposes of haematology analysis (Beckman Coulter UK Ltd, High Wycombe, UK).
• Maecker et al (BMC Immunology 2005, 6:13 ) discloses activated, fixed and cryopreserved whole blood samples for standardizing cytokine flow cytometry or intracellular cytokine staining.
• Cryopreserved cells also require specialised storage using e.g. liquid nitrogen and shipment on dry ice, thus increasing costs. There is also the risk of thawing and re-freezing during shipment, in the event of power failure to refrigeration devices, say, leading to deterioration of the material, so rendering it useless as a reference standard. Furthermore, cyropreserved cells must be carefully thawed to ensure consistency of responses. It has proven difficult to obtain consistent results between laboratories using this approach.
• the invention provides a method of preparing cellular reference material comprising the steps of: (a) providing a cell sample that has previously been isolated from a human or animal body, said cell sample comprising cells selected from the group consisting of: peripheral blood mononuclear cells (PBMC) (such as basophils, neutrophils, eosinophils, monoctytes, lymphocytes (B-lymphocytes and T-lymphocytes) and natural killer cells), thrombocytes and cell lines; (b) stimulating said cells to produce cytokines in the presence of a cytokine secretion inhibitor; (c) fixing said stimulated cells by addition of a fixative; (d) preserving said fixed stimulated cells by freeze drying.
• PBMC peripheral blood mononuclear cells
• cell lines we mean a cell culture capable of proliferation given appropriate fresh media and space, such cells being originally isolated from a human or animal body, and capable of cytokine production. Such cells may either produce cytokines inherently, or may be modified to include exogenous genetic material coding for, and leading to synthesis of, one or more cytokines, e.g. including but not restricted to TNF- ⁇ , IFN- ⁇ , IL-1 and IL-6.
• Exogenous DNA may be introduced into a recipient eukaryote cell by, say, micro-injection, electroporation, the use of calcium phosphate or a liposomal transfection reagent, and said genetic material may be subsequently integrated into the chromosomal DNA of said cells, or may remain present as e.g. a plasmid.
• a recipient eukaryote cell by, say, micro-injection, electroporation, the use of calcium phosphate or a liposomal transfection reagent, and said genetic material may be subsequently integrated into the chromosomal DNA of said cells, or may remain present as e.g. a plasmid.
• cytokines For cells that are capable of producing cytokines inherently, such cells may be modified, e.g. by the introduction of promoter sequences to upregulate cytokine production. Cells may be stimulated if required, e.g. by use of a mitogen, to produce cytok
• cytokine gene Transduction of a cytokine gene into neoplastic cells is known to elicit a strong inflammatory host reaction (see e.g. " The boosting effect of co-transduction with cytokine genes on cancer vaccine therapy using genetically modified dendritic cells expressing tumor-associated antigen", International journal of oncology; OJIMA Toshiyasu et al : ISSN 1019-6439 2006, vol. 28, no4, pp. 947-953 ). In vivo, this impairs tumour growth. In vitro, intra-cellular cytokines may be preserved according to the technique(s) described herein.
• Such cell lines may include cell lines derived from basophils, neutrophils, eosinophils, monoctytes, lymphocytes (B-lymphocytes and T-lymphocytes), natural killer cells (all peripheral blood mononuclear cells, PBMC) and thrombocytes, or cell lines such as HeLa, HL-60, A-549, Jurkat, LNCap and CAPAN-1 cells.
• the invention provides a method of preparing cellular reference material comprising the steps of: (a) providing a cell sample that has previously been isolated from a human or animal body, said cell sample comprising cells selected from the group consisting of: peripheral blood mononuclear cells (PBMC), and thrombocytes; (b) stimulating said cells to produce cytokines in the presence of a cytokine secretion inhibitor; (c) fixing said stimulated cells by addition of a fixative; (d) preserving said fixed stimulated cells by freeze drying.
• PBMC peripheral blood mononuclear cells
• thrombocytes thrombocytes
• the invention also provides a method of preparing cellular reference material comprising the steps of: (i) providing a mixed cell population that has previously been isolated from a human or animal body, said mixed cell population comprising a plurality of cell types selected from the group consisting of: peripheral blood mononuclear cells; thrombocytes; (ii) fractionating said mixed cell population to produce a plurality of fractions having distinct populations of cell types within each fraction; (iii) stimulating cells within one or more of said fractions to produce cytokines in the presence of a cytokine secretion inhibitor; (iv) fixing said cells within each fraction by addition of a fixative; (v) recombining a plurality of said fractions to produce a mixture of differentially-stimulated cells; (vi) preserving said mixture of differentially-stimulated cells by freeze drying.
• cytokine expression standards mimicking a particular pattern of cytokine expression in different cell types may be produced.
• Such cytokine expression standards would provide a benefit in both the clinical and research setting by assisting in both the diagnosis/prognosis of potential disease states and in the analysis of material/experimental outcomes within the research laboratory/empirical setting.
• said cell sample or population has been isolated from a mammalian body, and more preferably from a human body.
• said cells are stimulated by the addition of a mitogen.
• said mitogen comprises PMA (phorbol-12-myristate-13-acetate).
• said mitogen comprises Ionomycin. More preferably, said mitogen comprises a mixture of PMA (phorbol-12-myristate-13-acetate) and Ionomycin.
• said mitogen comprises a mixture of PHA (Phyto-hemagglutinin), IL-2 (interleukin-2) and co-stimulatory anti-human CD28 monoclonal antibody.
• said cytokine secretion inhibitor comprises Brefeldin A.
• said fixative comprises paraformaldehyde.
• said fixative comprises a mixture of paraformaldehyde and chromium chloride. Fixation has the benefit of suspending intra- and extra-cellular biological activity, including apoptosis, thus improving the biological and structural integrity of the cells during the freeze-drying process.
• the method further comprises the step of removing residual fixative after fixing said cells and before preserving said cells. This allows cells to be used immediately upon reconstitution without additional washing step to remove fixatives.
• One benefit of this step is that it minimises volumetric variation of cell numbers. Excess fixative can cause non-specific staining and prevent the release of cytokines in ELISPOT assays.
• the method further comprises the step of exposing said fixed cells to hypertonic conditions prior to preserving said cells.
• the method further comprises the step of adding a cryopreservative to said fixed cells prior to preserving said cells.
• a cryopreservative has the benefit of improving the structural integrity of the cells during the freeze-drying process.
• Also included in the scope of the invention is a method of preparing cellular reference material comprising the steps of: providing a plurality of cell samples that have previously been isolated from a plurality of individuals; carrying out a method described above, according to the first aspect of the invention, on each cell sample; and further comprising the step of combining fixed cells derived from a plurality of said samples prior to preserving said cells.
• Also included in the scope of the invention is a method of preparing cellular reference material comprising the steps of: providing a plurality of mixed cell populations that have previously been isolated from a plurality of individuals; carrying out a method described above, according to the second aspect of the invention, on each mixed cell population; and further comprising the step of combining fixed cells derived from a plurality of said mixed cell populations prior to preserving said cells.
• This has the added benefit of allowing the synthesis/production of mixed lymphocyte populations which both quantitively and qualitatively mimic the mixed lymphocyte populations found in various disease states. For example, with the onset of Graves disease, thyrocytes express cytokines they normally would not, such as TNF- ⁇ , IFN- ⁇ , IL-1 and IL-6.
• a method of preparing cellular reference material comprising the steps of: preparing unstimulated fixed cells according to the method steps of any preceding aspect in which the stimulation step (b) or (iii) is omitted and in which the preserving step (d) or (vi) is omitted; combining said unstimulated fixed cells with stimulated fixed cells prepared according to the method steps of any preceding aspect in which the preserving step (d) or (vi) is omitted; and preserving said combined cells by freeze drying or cryopreservation.
• This has the added benefit of providing mixed lymphocyte populations which mimic the non-diseased state as a comparator for disease-state populations, i.e. to act as a negative control.
• cryopreserved cells require specialised storage using e.g. liquid nitrogen and shipment on dry ice, thus increasing costs. There is also the risk of thawing and re-freezing during shipment, in the event of power failure to refrigeration devices, say, leading to deterioration of the material, so rendering it useless as a reference standard. Furthermore, cyropreserved cells must be carefully thawed to ensure consistency of responses. Freeze-drying therefore provides considerably more stable reference materials.
• "buffy coats” are obtained e.g from phlebotomised donors or a national blood collection agency.
• the buffy coat fraction of blood is that portion of blood that, following centrifugation, contains the majority of the white blood cells and platelets. All such obtained buffy coats will typically be tested to ensure they are negative for Treponema pallidum haem-agglutination test (“TPHA”), Hepatitis B surface antigent (“HBsAg”), anti Human Immunodeficiency Virus 1 (“anti-HIV1”), anti-Human Immunodeficiency Virus 2 (“anti-HIV 2”) and anti Hepatitis C Virus (“anti-HCV”) prior to use.
• TPHA Treponema pallidum haem-agglutination test
• HBsAg Hepatitis B surface antigent
• anti-HIV1 anti Human Immunodeficiency Virus 1
• anti-HIV 2 anti-Human Immunodeficiency Virus 2
• Residual erythrocytes contained within the buffy coats are removed by application of any standard, commercially available lymphocyte density gradient preparation, such as Lymphoprep, and peripheral blood mononuclear cells ("PBMC") are subsequently isolated/collected, again by application of a lymphocyte density gradient preparation.
• PBMC peripheral blood mononuclear cells
• the PBMC are then washed in a mammalian cell culture medium such as RPMI 1640 media.
• the PMBC are immuno-stimulated using T-cell stimulating mitogens, typically within an environment containing 4 - 6% carbon dioxide, and at a temperature of 36 - 38°C.
• T-cell stimulating mitogens typically within an environment containing 4 - 6% carbon dioxide, and at a temperature of 36 - 38°C.
• a combination of immuno-stimulants are used, said stimulants commonly comprising phorbol 12-myristate 13-acetate (PMA) (applied at a concentration of 0.01 - 0.03 ⁇ g per ml, say 0.02 ⁇ g per ml) and Ionomycin (applied at a concentration of 0.125 - 0.165 ⁇ g per mg, say 0.145 ⁇ g per mg).
• the stimulation technique is suitable for cytokine-production, and if immuno-stimulated for 4 - 6 hours, cytokine production within the cells PMBC is detectable.
• the PMBC are simultaneously combined with a commercially available extra-cellular protein transport inhibitor, such as Brefeldin A or Monensin, which results in cytokine accumulation within the said PMBC.
• a commercially available extra-cellular protein transport inhibitor such as Brefeldin A or Monensin
• Brefeldin A it would commonly be applied at a concentration of 9.0 - 11.0 ⁇ g per ml, say 10 ⁇ g per ml.
• the PBMC are washed in a combination of foetal calf serum (“FCS”) and phosphate buffered saline (“PBS”), the FCS being used typically at a concentration of 9 - 12% v/v, say 10% and the PBS typically being double strength.
• FCS foetal calf serum
• PBS phosphate buffered saline
• the washed PBMS are then re-suspended in a buffered culture medium (again comprising double strength PBS and 9 - 12% FCS), with the addition of 0.1-20% (v/v) of a commercially available transport fixative containing a combination of paraformaldehyde (0.1-0.2% w/v) and chromium chloride (0.5% w/v) in 0.85% (w/v) PBS.
• a commercially available transport fixative containing a combination of paraformaldehyde (0.1-0.2% w/v) and chromium chloride (0.5% w/v) in 0.85% (w/v) PBS.
• TransFix An example of one such fixative is known by the trade name "TransFix”.
• the buffered culture medium and fixative is applied to the said PBMC at a rate of 8 x 10 6 PBMC per ml.
• PBMC are fixed immediately following isolation in paraformaldehyde and chromium chloride, as described above, and stored until required at +4°C.
• the PBMC are subsequently washed with chilled (+4°C) freeze drying buffer (a cryoprotectant).
• a typical buffer would comprise a double volume of 10% protein, typically, foetal calf serum or albumin in double strength PBS.
• the use of a freeze drying buffer improves PBS stability during the subsequent freeze drying process.
• These cells may then be stored at chill temperatures (typically +4°C) before freeze-drying.
• chill temperatures typically +4°C
• the shelves of a freeze dryer are pre-cooled in order to maintain a temperature of +4°C during the loading process.
• cells from a plurality of donors may be mixed together to increase the total volume of reference material so produced.
• the process of fixing allows cells from different donors to be mixed together without causing lymphocyte cross-reactions.
• fixed stimulated cells may be mixed with fixed, unstimulated cells (which can also be used as negative controls) in order to produce a cell population demonstrating typical levels of cytokine stimulation or for a minimum potency positive control.
• the stimulated (or unstimulated), fixed cells are loaded into aliquots/capped vials.
• a filling machine such as one sold under the trade name Paxal, is suitable for large scale aliquoting.
• the PBMC are freeze-dried, typically over a 5-day cycle. Residual moisture content after freeze drying is typically 0.35%.
• the PBMC Once fixative has been washed from the stimulated cells, the PBMC begin to leak cytokines, albeit slowly at low temperatures. Therefore, loading into aliquots and freeze drying of PBMC is performed within a few hours of fixative removal.
• the lyophilised reference materials may be stored for extended periods of time without degradation.
• the freeze-dried samples may be reconstituted in twice the starting volume of distilled water. This typically gives cells in single-strength phosphate buffered saline.
• fractions of cells rich in a particular cell type may be isolated from a mixed population of PMBC and subjected to the stimulation and fixing regime described above before being re-combined with fixed, unstimulated cells.
• cell populations with different levels of cytokines in specific cell types may be produced to mimic particular disease states.
• Particular cell types may be isolated by a number of means such as flow cytometry and cell-sorting, or by the use of antibody-linked magnetic beads, available commercially.
• Such separation techniques may be used to isolate e.g. B cells, monocytes, natural killer cells, neutrophils, platelets, etc., as well as particular subsets such as CD4 and CD8 T-cells.
• populations of differentially-stimulated cells may be diluted with unstimulated, fixed cells to produce cell populations that are a closer mimic to those found in particular disease states.
• Cell reference standards produced according to methods of the invention may be reconstituted by addition of water, and used in cytokine assays.
• the cells retain surface antigens, as well as intracellular cytokines. They may be subjected, therefore, to staining techniques used in the art and used as reference standards for e.g. flow cytometry. Furthermore, the cells remain intact, and yet capable of releasing cytokines into their immediate environment, especially when held at elevated temperatures, e.g. 37°C, as commonly used in ELISPOT assays. They may therefore be used as reference standards in this type of assay.
• freeze-dried cells produced by the methods described herein can also find use as vaccines as the cells' surface antigens are maintained in an intact state by the fixing and freeze drying process.

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